Ionotropic glutamate receptors (iGluRs) are membrane proteins which act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The 7 gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits which assemble to form 3 major functional families named after the ligands which were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate and NMDA. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of iGluR activity mediates multiple neurological and psychiatric diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of GluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design electrophysiological and biochemical experiments to define the allosteric mechanisms underlying ligand recognition and the gating of ion channel activity. This information will allow the development of subtype selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine which plays a key role in brain function.[unreadable] [unreadable] Crystallographic and functional analysis of glutamate receptor ligand complexes[unreadable] Alushin and Mayer in collaboration with Jane[unreadable] [unreadable] High resolution crystal structures were solved for four GluR5 subtype selective antagonist complexes; three are derivatives of willardiine based compounds for which we solved the 1st GluR5 antagonist complex in 2006, and one is for a decahydroisoquinoline which has potential as an analgesic and drug to reduce migraine. The structures reinforce the idea that even when bound with competitive antagonists glutamate receptors can sample a range of conformational space, similar in principle to the variation in domain closure observed for partial agonists, with the key difference that the difference in domain closure for individual antagonists does not cross the threshold necessary to trigger ion channel gating. One of the structures revealed a novel protein ligand interaction, named a halogen bond, formed by a contact between a carboxylate side chain and a ligand bromine atom.[unreadable] [unreadable] Crystallographic and functional analysis of allosteric ion binding sites[unreadable] Plested and Mayer in collaboration with Biggin [unreadable] [unreadable] Kainate subtype glutamate receptors are strongly modulated by monovalent anions and cations and in the absence of either chloride or sodium the receptors become non functional. A combined experimental approach using crystallography and patch clamp recording was used to identify the binding site for anions. The chloride ion binds in the dimer interface between two subunits and acts as electrostatic glue which helps to stabilize dimer assemblies in their active conformation. In the absence of chloride the dimers dissociate, and the receptor desensitizes. Mutations which disrupt chloride binding have the same effect in functional experiments. These results reveal that instead of acting in a modulatory, allosteric manner, anions are instead essential structural components of the receptor in its active conformation. In ongoing work using the same approach, we are working solve the structure of the cation binding site.[unreadable] [unreadable] Structural analysis of NR3 ligand binding selectivity[unreadable] Yao and Mayer[unreadable] The NMDA receptor NR3A subunit is expressed widely in the developing CNS of mammals. Co-assembly of NR3A with NR1 and NR2 modifies NMDA receptor-mediated responses, reducing calcium permeability. In previous work we characterized the ligand binding properties of NR3A using a highly purified water soluble NR3A ligand binding domain. High resolution crystal structures have now been solved for NR3A complexes with glycine, D-serine and ACPC, and for NR3B complexes with glycine and D-serine. These reveal that despite the substitution by methionine of a large tryptophan residue, which in the NR1 subunit fills up the binding pocket, preventing the binding of glutamate, the binding pocket of NR1, NR3A and NR3B is unusually small compared to other glutamate receptor subtypes which bind glutamate, indicating that steric occlusion is a common principle for achieving glycine selectivity. In ongoing work a series of mutations based on the crystal structures for NR1 and NR3 subunits are being tested to test the hypothesis that subunit specific differences in ligand affinity result in part from formation of closed cleft, i.e. active conformations